1. Field of the Invention
The present invention relates to an isolated and purified bifunctional protein from carrots (Daucus carota L. cv Danvers) with aspartokinase and homoserine dehydrogenase activities. The invention further relates to a nucleic acid fragment encoding a bifunctional protein with aspartokinase and homoserine dehydrogenase activities.
2. Description of the Background Art
Plants can convert asparate to the amino acids methionine, threonine, lysine and isoleucine (J. Bryan, Biochemistry of Plants, (B. Miflin ed.) Academic Press, New York, pp. 403-452 (1980)). As these amino acids are essential in the diets of many animals, there is much interest in understanding the control mechanisms that determine the quantity of these essential amino acids in food sources. Enzymes control the pathways leading to the synthesis of the essential amino acids methionine, threonine, lysine and isoleucine. The isolation of clones of the enzyme genes from plants would enable one to determine the relationship between various forms of the enzymes, the number of genes involved, and the regulation of the pathway. Knowledge gained from the study of the amino acid pathway genes would allow the engineering of the pathway to alter the amino acid pool composition of plants used as protein sources.
Aspartokinase (AK.2) (E.C. 2.7.2.4) and homoserine dehydrogenase (HSDH) (E.C.1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, methionine, and threonine from aspartate. AK.2 catalyzes the phosphorylation of aspartate to .beta.-aspartyl phosphate. It is the first enzyme of the pathway leading to the synthesis of the essential amino acids lysine, threonine, methionine and isoleucine in plants. .beta.-aspartyl phosphate is converted to aspartate semialdehyde, which can either be used to make lysine or it can be reduced by the enzyme homoserine dehydrogenase (HSDH) to homoserine. Through further enzymatic steps homoserine is converted first to phosphohomoserine and eventually to threonine and isoleucine or methionine.
In higher plants there are commonly at least two forms of AK which are differentially feedback inhibited by the end products lysine and threonine (See H. Davies et al., Plant Physiol 62: 536-541 (1978); B. Matthews et al., Planta 141: 315-321 (1978); B. Matthews et al., Z Naturforsch 346: 1177-1185 (1979); B. Matthews et al., Z Pflanzenphysiol 96: 453-463 (1980) and K. Sakano et al., Plant Physiol 61: 115-118 (1978)). HSDH.2 (EC 1.1.1.3) catalyzes the reversible conversion of aspartate semialdehyde to homoserine and is at the branch point leading to threonine, methionine and isoleucine synthesis.
Regulation of carrot (Daucus carota) AK and HSDH activities from roots and cell suspension cultures has been studied extensively (See H. Davies, supra; B. Matthews (1978, 1979, 1980) supra; J. Relton et al., Biochim Biophys Acta 953: 48-60 (1988); K. Sakano Plant Physiol 63: 583-585 (1979); and K. Sakano, supra). HSDH has been purified to apparent homogeneity and characterized (B. Matthews et al., Plant Physiol 91: 1569-1574 (1989)). Two forms of HSDH have been identified in vitro: one sensitive to threonine inhibition and one insensitive. Carrot HSDH activity reversibility converts between a threonine-insensitive form in the presence of K+ and a threonine-sensitive form in the presence of threonine which possess distinctly different electrophoretic mobilities on PAGE gels stained for enzymatic activity. Three forms of aspartate kinase have been isolated from carrot: form I is strongly inhibited by lysine, form II is strongly inhibited by threonine, and form III is partially inhibited by both. The relationship between these three forms is not yet defined. Antibody to this HSDH has been examined for specificity and cross reactivity with soybean and E. coli (F. Turano et al., Plant Physiol 92: 395-400 (1990)).
These biosynthetic pathways in plants are similar to pathways found in bacteria (G. Cohen et al., Cellular and Molecular Biology (F. Neidhardt ed.) American Society for Microbiology, Washington, D.C., pp. 429-444 (1987); and G. Cohen, Amino Acids: Biosynthesis and Genetic Regulation (K. Hermann and R. Somerville, eds.) Addison-Wesley, Reading, pp. 147-171 (1983)). Many of the bacterial genes that code for the enzymes of the aspartate pathway have been cloned and sequenced (G. Cohen (1987), supra). Only one of the plant genes in this pathway has been isolated (dihydrodipicolinate synthase (T. Kaneko, J Biol Chem 265: 17451-55 (1990)), which catalyzes the first reaction specific to lysine synthesis).
In E. coli there are three genes coding for aspartate kinase and/or homoserine dehydrogenase. One, lysC, codes for a lysine-sensitive aspartate kinass (AKIII), is regulated by lysine and does not contain HSDH activity. The other two genes code for bifunctional AK-HSDH proteins (G. Cohen (1983), supra). ThrA is repressed by threonine and isoleucine and the enzymatic activity of AKI-HSDHI is inhibited by threonine. MetL is repressed by methionine, but the protein AKII-HSDHII is not responsive to end product inhibition. It is not known if the multiple enzyme forms in plants are encoded by separate genes or if these genes are subject to transcriptional or translational regulation.
In Bacillus subtilis (R. Bondaryk et al., J Biol Chem 10: 585-591 (1985); and N. Chen et al., J Biol Chem 262: 8787-8798 (1987)), Brevibacterium lactofermentum (Mateos et al., Nucleic Acids Research 15:10598 (1987)), Rhodospirillum rubrum (P. Datta J Biol Chem 245: 5779-5787 (1970)) and Saccharomyces cerevisiae (J. Rafalski et al., J Biol Chem 263: 2146-2151 (1988)) HSDH and AK activities reside on separate proteins encoded by separate genes.
Enzymes involved in the synthesis of the aspartate family of amino acids appear to be relatively low in abundance. Homoserine dehydrogenase has been purified to homogeneity from maize (T. Walter et al., J Biol Chem 254: 1349-1355 (1979)) and carrot (B. Matthews (1989), supra), while aspartokinase has been purified to homogeneity from maize (S. Dotson et al., Plant Physiol 91: 1602-1608 (1989)) and partially purified from carrot (B. Matthews (1978, 1979), supra). There have been no indications in the literature that these two enzyme functions reside on the same protein in plants. In most of the plant species studied multiple forms of AK and HSDH have been identified in vitro (J. Bryan, supra). These forms are distinguished by their sensitivity to feedback inhibition (in particular by threonine and lysine) and by their molecular weight and subunit composition. Because there are AK activities sensitive to lysine and threonine but HSDH activity is sensitive only to threonine, a common peptide was not suspected. Although HSDH activity is associated with both lysine- and threonine-sensitive AK, the ratio of activities is variable for reasons unknown at this time. In E. coli AKI-HSDHI both enzymatic activities are inhibited by threonine. The E. coli AKIII is inhibited by lysine or threonine. Because of these E. coli examples, an aspartokinase sensitive to lysine and associated with threonine-sensitive homoserine dehydrogenase appears to be inconsistent.
Other factors have prevented detection of these coincident activities. It has been observed that both AK and HSDH eluted off the gel filtration column at the same location but it was assumed that it was a simple case of coelution of two large, similarly-sized proteins. The purification protocol (Matthews (1989), supra) for HSDH had already been established before the protocol for AK. In the protocol for purification of HSDH from carrot suspension culture cells (Matthews (1989), supra), a heat denaturation step is utilized and activity of AK is lost after the heat denaturation step. Even though AK activity of the E. coli bifunctional AKI-HSDHI is lost after heating, the comparison was not made because a bifunctional protein in plants was not suspected.
Furthermore, in contrast to E. coli, not all bacteria have bifunctional AK-HSDHs. In Brevibacterium lactofermentum separate genes encode separate AK and HSDH proteins. The B. subtilis gene possesses two initiation sites to produce AKI and a truncated, but functional AKII protein (N. Chen, supra). In yeast, a gene encoding AK also has been identified (J. Rafalski, supra); this gene does not appear to encode HSDH. HSDH has been extensively examined in Rhodospirillum rubrum (P. Datta et al., J Biol Chem 240: 3023-3033 (1965); C. Epstein et al., Eur J Biochem 82: 453-461 (1978); and P. Datta (1970), supra) but there are no reports in the literature that this protein also contains AK activity.